朱甫祥，刘泽隆，缪静，屈慧鸽，迟晓艳. Cys突变体凝血VIII因子的蛋白质反式剪接J. 药学学报, 2012,47(6): 734-738.
引用本文: 朱甫祥，刘泽隆，缪静，屈慧鸽，迟晓艳. Cys突变体凝血VIII因子的蛋白质反式剪接J. 药学学报, 2012,47(6): 734-738.
shu
ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan. Trans-splicing of Cys mutated coagulation factor VIIIJ. 药学学报, 2012,47(6): 734-738.
Citation: ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan. Trans-splicing of Cys mutated coagulation factor VIIIJ. 药学学报, 2012,47(6): 734-738.
shu

Cys突变体凝血VIII因子的蛋白质反式剪接

Trans-splicing of Cys mutated coagulation factor VIII

    摘要
  • 摘要:

    为改善蛋白质反式剪接效率, 将B-区缺失型FVIII (BDD-FVIII) 重链的Tyr664和轻链的Thr1826突变为Cys, 双载体共转COS-7细胞, 观察了细胞内蛋白质剪接、二硫键形成和细胞培养上清液中分泌的剪接BDD-FVIII量和活性。Western blotting检测结果显示, Cys突变可在细胞内形成链间二硫键, 剪接BDD-FVIII蛋白量明显增加; 双夹心ELISA检测分泌的剪接BDD-FVIII为 (128 ± 24) ng·mL−1, 明显高于对照 (89 ± 15) ng·mL−1; Coatest法检测结果显示, 细胞分泌的凝血活性为 (0.94 ± 0.08) u·mL−1, 也明显高于对照 (0.62 ± 0.15) u·mL−1。结果表明, 链间二硫键形成可显著提高基于蛋白质剪接的双载体转BDD-FVIII基因作用, 为进一步动物体内实验提供了依据。

     

    Abstract:

    To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr664 in heavy chain and at Thr1826 in light chain of B-domain-deleted FVIII (BDD-FVIII).  By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed.  The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 ± 24 ng·mL−1) compared to control (89 ± 15 ng·mL−1), assayed by a sandwich ELISA.  A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 ± 0.08 u·mL−1) compared to that of control (0.62 ± 0.15 u·mL−1).  It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

     

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